Method of using pomegranate extracts for increasing prostate specific antigen doubling time

ABSTRACT

Pomegranate juice consumption by a patient may be used to treat prostate cancer. Prior to the present invention, it has been observed that various pomegranate compositions have cancer chemopreventive properties on various in vitro treatment of human prostate cancer cell lines and in vivo treatment of human prostate cancer cell lines xenografted in athymic nude mice. Until the discovery of the present invention, it was not known if there is an efficacy for treating prostate cancer in human patient by any route of administration. The present invention provides a method of treating a subject with prostate cancer a composition comprising a therapeutically effective amount of an extract from pomegranate fruit. The extract of pomegranate may be a juice extract of pomegranate, an extract from inner or outer peel of pomegranate, or mixture thereof. Pomegranate juice may be used to treat subjects with rising serum PSA after prostate surgery or radiotherapy.

This application claims benefit of U.S. Provisional Patent ApplicationSer. No. 60/782,437 filed Mar. 15, 2006, the specification of which ishereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to pomegranate extracts andmethods of using thereof for treating prostate cancer in a way thatincreases prostate specific antigen doubling time.

2. Description of the Related Art

Phytochemicals in edible plants can have cancer preventative benefitsthrough antioxidation and via gene-nutrient interactions. Pomegranatejuice has been shown to be a rich source of polyphenolic flavonoids.Pomgranate juice possesses impressive antioxidative properties due toits high flavonoids content, mainly the water soluble tannins andproanthocyanins.

Prostate cancer is the most common invasive malignancy and the secondleading cause of cancer-related deaths among U.S. males, with a similartrend in many Western countries. Biological aggressiveness of prostatecancer is directly related to tumor volume, and tumor volume isproportional to serum prostate specific antigen (PSA). Age is the mostcommon risk factor, with nearly 70 percent of prostate cancer casesoccurring in men age 65 and older. The higher a man's PSA level, themore likely it is that cancer is present. However, recent research foundprostate cancer in men with PSA levels below 4.0 ng/ml. Most men with anelevated PSA test turn out not to have cancer; only 25 to 30 percent ofmen who have a biopsy due to elevated PSA levels actually have prostatecancer. A prostate biopsy is the main method used to further diagnoseprostate cancer.

Many patients who undergo surgery or radiation therapy to treatlocalized prostate cancer experience an increase in PSA level aftertreatment. One of methods being used is measurement of PSA velocity,which is based on changes in PSA levels over time. A sharp rise in thePSA level raises the suspicion of cancer. The rate at which the PSAlevel is rising for patient, after radical prostatectomy or radiationtherapy for localized prostate cancer, is known to correlate withaggressiveness and growth rate of the cancer thus a rapidly rising PSAis correlated with worse patient outcomes and vice-versa. In a study ofuntreated patients with prostatic carcinomas, patients with shorterdoubling times were found with higher clinical stages and worsehistological grades whereas patients with longer doubling time werefound to have more favorable outcome with low grade tumors on deferredtreatment. In men with prostate cancer that has been treated withcombined hormone and radiation therapy, a post treatment PSA doublingtime of < or =8 months is associated with worse clinical outcomes andmay be an early surrogate marker for decreased survival.

Various studies described in vitro cancer chemopreventive properties,including anti-proliferative and pro-apoptotic effects, of differentpomegranate compositions on various cancer cell lines. Several studiesinvestigated effects of pomegranate compositions on prostate cancer celllines. The Lansky et al. studies observed different pomegranate mixtureon inhibition of human prostatic adenocarcinoma PC-3 invasion andproliferation across Matrigel and inhibition of phospholipase A-2expression associated with invasive potential. The Malik et al. studydescribed pomegranate fruit extract on inhibition of cell growthfollowed by apoptosis of PC-3 through modulations in the cyclin kinaseinhibitor-cyclin-cyclin dependent kinase (cdk) signal transductionpathways. The Malik et al. study also described pomegranate fruitextract administration to athymic nude mice implanted withandrogen-sensitive human prostatic CWR22Rv1 cells resulted in asignificant inhibition in tumor growth concomitant with a significantdecrease in serum prostate-specific antigen levels. The results ofpomegranate in vitro antitumor activity on human prostatic cancer celllines and in vivo xenograft of CWR22Rv1 cell only suggest possiblecancer-chemopreventive as well as cancer-chemotherapeutic effects for invivo treatment of prostate cancer in human.

The Albrecht et al. study observed in vitro antitumor activities ofpomegranate extracts on cell proliferation, cell cycle distribution,apoptosis, gene regulation, and tumor growth for PC-3, human prostaticcarcinoma LNCaP, and human prostatic carcinoma DU145 human cancer celllines whereas normal prostate epithelial cells (hPrEC) weresignificantly less negatively affected. The Albrecht et al. study alsoobserved potent inhibition on tumor growth for in vivo xenograft of PC-3in athymic nude mice injected subcutaneously with pomegranate extracts.The observation of subcutaneous injection of pomegranate extract on thegrowth inhibition of in vivo xenograft of PC-3 in nude mice does notvalidate in vivo treatment of prostate cancer in human subjects becauseit is not known if any administration of pomegranate extract have anantitumor activity on prostate cancer in human.

In vitro tests and in vivo animal tests are necessary to evaluatepotential therapeutic effect of pomegranate compositions prior toclinical evaluations. The translation of in vitro and in vivo studiesfrom the laboratory into the clinical trial in human is necessary toobtain and validate anticancer therapeutics for efficacy and tolerationas numerous human trials of anticancer drugs have not advanced to adefinitive assessment of clinical efficacy in a clinical trial. Prior tothe present invention, none of prior art studies have advancedpomegranate juice to a phase II trial for treating prostate cancer. Thepurpose of a phase II trial of a new anticancer drug is to determinewhether the drug has sufficient activity against a specified type oftumor to warrant its further development.

BRIEF SUMMARY OF THE INVENTION

The present invention is based on the discovery that pomegranate juiceconsumption by a patient may be used to treat prostate cancer. Prior tothe present invention, it has been observed that various pomegranatecompositions have cancer chemopreventive properties on various in vitrotreatment of human prostate cancer cell lines and in vivo treatment ofhuman prostate cancer cell lines xenografted in athymic nude mice. Untilthe discovery of the present invention, it was not known if there is anefficacy for treating prostate cancer in human patient by any route ofadministration.

The present invention provides a method of treating a subject withprostate cancer a composition comprising a therapeutically effectiveamount of an extract from pomegranate fruit. The extract of pomegranatemay be a juice extract of pomegranate, an extract from inner or outerpeel of pomegranate, or mixture thereof.

According to an embodiment of the present invention, pomegranate juicemay be used to treat subjects with rising serum PSA after prostatesurgery or radiotherapy. The present invention describes efficacy ofpomegranate juice treatment, which shows in vivo effect on increasingPSA doubling time for subjects with prostate cancer post-surgery orradiation with rising PSA level. The cancer chemotherapeutic propertiesof pomegranate juice is further evidenced by another embodiment of thepresent invention which provides for a method in that serum derived fromsubjects on daily pomegranate juice administration for in vitrotreatment of human prostate cancer cell line which showanti-proliferative and pro-apoptotic actions. Other objects, featuresand advantages of the present invention will become apparent to thoseskilled in the art from the detailed description. It is to beunderstood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the presentinvention, are given by way of illustration and not limitation. Manychanges and modifications within the scope of the present invention maybe made without departing from the spirit thereof, and the inventionincludes all such modifications and equivalents thereof.

DESCRIPTION OF THE FIGURES

FIG. 1 shows LN(PSA) plotted over time in 1 patient before and duringpomegranate juice treatment demonstrating a statistically and clinicallysignificant prolongation of the PSADT with pomegranate juice treatment.

FIG. 2 shows the effect of pre and post treatment serum on growthinhibition of LNCaP cells after 96 hours.

FIG. 3 shows LC-MS analysis of urines, pre and post treatment, obtainedfrom a patient orally administered with pomegranate juice. The relativeintensities are given by the figures in the top right hand corner ofeach chromatogram.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention provides a method for treating apatient with prostate cancer. The method comprises the step ofadministering to the patient a composition comprising a therapeuticallyeffective amount of an extract from pomegranate.

For the purpose of the present invention, an extract from pomegranatemay be an extract from the whole pomegranate fruit or from anyconstituents of pomegranate fruit. Examples of constituents ofpomegranate fruit that may be used to make the extract of the presentinvention include, but are not limited to, juice, seed, and the innerand outer peel of pomegranate fruit. In one embodiment of the presentinvention, the extract is the juice extract of whole pomegranate fruit.In another embodiment of the present invention, the extract is from theinner or outer peel of pomegranate fruit. In a further embodiment of thepresent invention, the extract may be a mixture of two or more extractsof the whole pomegranate or any constituents of pomegranate. The term“phytochemicals” generally refers collectively to compounds which arenaturally-occurring in the pomegranate and to reaction products andmetabolites of these compounds, which are considered to have abeneficial effect on the human health. Examples of such phytochemicalsinclude, but are not limited to polyphenols, estrogens andphytoestrogens. The term “polyphenols” refers generally to a family ofnaturally-occurring compounds in the pomegranate and includes phenolsand polyphenols. Phenols are a class of chemical compounds consisting ofa single phenol unit in their structure. Although similar to alcohols,phenols have unique properties including relatively higher acidities dueto the aromatic ring tightly coupled to the oxygen and a relativelyloose bond between the oxygen and the hydrogen. Examples of phenoliccompounds within this group include ellagic acid and gallic acid.Polyphenols are a group of compounds, characterized by the presence ofmore than one phenolic group. Polyphenols include tannins (e.g.,ellagitannins and gallotannins), flavonoids (e.g., anthocyanins andisoflavones) and stilbenes (e.g., resveratrol). The term “pomegranatejuice” refers to the juice that is substantially obtained from the arilsof the pomegranate. The term “pomegranate solids” refers to any one or acombination of the pericarp, the inner membrane and seeds of apomegranate.

Methods of making the extract, including the juice from wholepomegranate fruits are described in U.S. Pat. No. 6,977,089 entitled“METHODS OF USING POMEGRANATE EXTRACTS FOR CAUSING REGRESSION IN LESIONSDUE TO ARTERIOSCLEROSIS” and in U.S. patent application Ser. No.11/137,248 entitled “PROCESSES FOR EXTRACTING PHYTOCHEMICALS FROMPOMEGRANATE SOLIDS AND COMPOSITIONS AND METHODS OF USE THEREOF” both ofwhich are incorporated herein by reference. In general, any methods thatmay produce pomegranate extract and juice that naturally occurs inpomegranate may be used. For the purpose of the present invention, thejuice may be concentrated or diluted from its natural concentration. Thejuice may also be mixed with extracts of other constituents ofpomegranate to increase effectiveness.

Extracts from the constituents of pomegranate, i.e., seeds or the inneror outer peel, may also be used alone or in combination with juice. Forexample, the seeds or the inner or outer peel of pomegranate may bediluted in water and the extract may be made by crushing, squeezing, orextensive vortexing. The insoluble materials of the extract may beseparated from the soluble supernatant of the extract. Preferably, thesupernatant of the extract is used for the purpose of the presentinvention, although any oily, lipidic fraction of the extract may alsobe used. The extract from constituents of pomegranate may beconcentrated or diluted, or mixed with each other or with pomegranatejuice extract.

In accordance with one embodiment of the present invention, the extractof the present invention may be prepared by a process including thesteps of: (a) crushing and squeezing the whole fruits of thepomegranate, including the inner and outer peels and the seeds, to yielda juice component and an insoluble by-product component, and (b)separating the juice component from the insoluble by-product component.The juice component may be used as a juice extract of the presentinvention. The insoluble by-product component may be resuspended in anaqueous medium, such as, but not limited to, water or alcohol, and befurther crushed, squeezed, and mixed to yield a soluble portion and aninsoluble portion. Then the soluble portion may be separated from theinsoluble portion to produce the extract of the constituents of thepresent invention. Alternatively, the soluble portion may be combinedwith the juice extract to produce the extract of the present invention.

In one embodiment of the present invention, the whole fruit of thepomegranate may be enzymatically treated to improve extraction andfiltration. For example, pectinase may be used to treat the whole fruitto prevent the formation of pectin gels. Other enzymes known in the artmay also be used as long as they can improve extraction and filtrationof the extract of the present invention.

The extract of pomegranate of the present invention may be in a liquidor solid form. In accordance with one embodiment of the presentinvention, a solid form of the extract may be made by lyophilizing theliquid extract of the present invention. Alternatively, the constituentsof the pomegranate, such as seeds, inner or outer peels, or anyinsoluble portion discussed above, may be processed directly to form thesolid form of the extract of the present invention. For example, theconstituents of the pomegranate may be dried, and “processed into powderor pill forms to be used directly as the solid form of the extract ofthe present invention.

In one embodiment of the invention pomegranate extract is prepared usingthe following method. Any one or a combination of the pericarp, innermembrane and seeds are selected and a mixture is formed comprising thepomegranate solids and an aqueous solution. The mixture is then heatedto about 60° F. to 210° F., preferably of about 85° F. to 185° F. andoptimally of about 110° F. to 160° F. Enzymes are added to the mixturein an amount sufficient to at least partially degrade the pomegranatesolids and liberate phytochemicals from the plant tissues and/or cells.Once liberated, the phytochemicals may react and/or polymerize to createnew phytochemical compounds or reaction products. The residual insolublesolid materials are removed from the mixture to provide an extractcontaining phytochemicals. In another embodiment, extracts containingphytochemicals from a pomegranate are provided and used for purposes ofthe treatment described herein. Such extracts are characterized by asignificantly higher total polyphenol content, particularly of the highmolecular weight polyphenol (e.g., punicalagin), than is found inpomegranate juice alone. Such extracts may be obtained from the methodsdisclosed herein.

In a further embodiment of the invention, food products and beveragesare provided comprising the extract containing phytochemicals from apomegranate. In yet a further preferred embodiment, compositionscomprising the extract containing phytochemicals from a pomegranate areprovided. Such compositions may be in form of tablets, suspensions,implants, solutions, emulsions, capsules, powders, syrups, liquidcompositions, ointments, lotions, creams, pastes, and gels. Suchcompositions may also be in form of pharmaceutical preparations,nutritional supplements, vitamin supplements, food additives, and foodsupplements.

In a further embodiment of the invention, compositions containing theextract and the pomegranate juice are provided. The combination of theextract and pomegranate juice not only produces a composition having ahigher total polyphenol content, as compared to the pomegranate juicealone, but it also provides the broad spectrum of the differentpolyphenols which predominate the pomegranate juice and extract. Inanother preferred embodiment, methods are provided for preventing orameliorating disease conditions in a subject by administering to thesubject an effective amount of the composition suitable for use as apharmaceutical or nutritional preparation. Such disease conditionsinclude polyphenol-mediated diseases and cancer.

In yet another embodiment, methods are provided for modulating thegrowth and progression of cancerous cells, the methods comprisingselecting a subject having cancerous cell growth and administering tothe subject an effective amount of the composition containing theextract. In yet a further embodiment, methods are provided forpreventing or slowing increases in the Prostate Specific Antigen (PSA)levels in a subject having prostate cancer. The method comprisesselecting a subject having prostate cancer and administering to thesubject an effective amount of the composition containing the extract.

Compositions used in the various embodiments of the invention describedherein may be a variety of kinds, including, but not limited to,nutritional supplements, pharmaceutical preparations, vitaminsupplements, food additives, or foods supplements. Compositions of thepresent invention may be in convenient dosage forms, including, but notlimited to, tablets, suspensions, implants, solutions, emulsions,capsules, powders, syrups, liquid compositions, ointments, lotions,creams, pastes, gels, or the like.

Compositions of the present invention may include a carrier. Dependingon the kind of compositions of the present invention, a carrier may be adietary suitable carrier or a pharmaceutically acceptable carrier, aslong as it is compatible with the particular kind of compositions of thepresent invention. Examples of a dietary suitable carrier include, butare not limited to, dietary suitable excipients, diluents, and carriers.Examples of a pharmaceutically acceptable carrier include, but are notlimited to, biocompatible vehicles, adjuvants, additives, and diluentsto achieve a composition usable as a dosage form. As used herein, theterms “pharmaceutically acceptable,” “physiologically tolerable,” andgrammatical variations thereof, as they refer to compositions, carriers,diluents, and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a mammal without theproduction of undesirable physiological effects.

The compositions of the present invention may be used alone or incombination with other biologically active ingredients. A composition ofthe present invention, alone or in combination with other activeingredients, may be administered to a subject in a single dose ormultiple doses over a period of time, generally by oral administration.Various administration patterns will be apparent to those skilled in theart. The dosage ranges for the administration of the compositions of thepresent invention are those large enough to produce the desired effect.The dosage should not be so large as to cause any adverse side effects,such as unwanted cross-reactions and the like. Generally, the dosagewill vary with the age, weight, sex, condition, and extent of acondition in a subject, and the intended purpose. The dosage can beadjusted in the event of any counter indications, tolerance, or similarconditions. Those of skill in the art can readily evaluate such factorsand, based on this information, determine the particular effectiveconcentration of a composition of the present invention to be used foran intended purpose.

In one embodiment of the present invention, a composition contains theextract of pomegranate in a dosage unit in an amount that contains atleast 30 to 3000 μmols per dosage unit of polyphenols. For the purposeof the present invention, polyphenols are those naturally present in theextract of pomegranate. It should be appreciated that polyphenols areused herein as a measurement marker for the amount of extract that needsto be used in each dosage unit. They are not used herein as anindication that they are the active, or the only active, ingredients ofthe extract. In fact, it is possible that something else, or the synergyof polyphenols and other components of an extract of the presentinvention, may be responsible for the activities of the extract.

The term “dosage unit” as used herein refers to physically discreteunits suitable as unitary dosages for animals, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect in association with the required diluent,e.g., a carrier or vehicle. The specifications for the unit dose of thisinvention are dictated by and are directly dependent on (a) the uniquecharacteristics of the active material and (b) the limitations inherentin the art of compounding such active material for therapeutical use inanimals.

The term “therapeutically effective amount” as used herein means thatthe amount of the extract of the present invention contained in thecomposition administered is of sufficient quantity to achieve theintended purpose, such as, in this case, to treat prostate cancer in thepatient. For the purpose of the present invention, treatment of prostatecancer may be measured by the increase in the serum prostate specificantigen doubling time (PSADT). For example, digital rectal exam,cystoscopy, transrectal ultrasonography, or prostate biopsy methods maybe used to measure stages or grades of prostate cancer.

Accordingly, by determining the increase of prostate specific antigendoubling time in a patient, one can readily determine whether the amountof the extract of the present invention is therapeutically effective. Inone embodiment, the therapeutically effective amount of the extract ofthe present invention contains at least 30 to 3000 μmols of polyphenolsnaturally occurring in a pomegranate fruit. Again, it should beappreciated that the polyphenols are used herein as a measurement markerfor the concentration of the extract of the present invention. Inanother embodiment, the composition contains one glass of juice extractof the present invention.

The method of the present invention may be used to treat patients withprostate cancer, including patients after prostatectomy, radiotherapy,cryosurgery, or hormonal therapy. In addition, the methods of thepresent invention may be used to treat in vitro human prostate cancercell lines, particularly to treat LNCaP, PC-3, CWR22Rv1, or DU145 cellsby promoting apoptosis and/or inhibiting proliferation. Furthermore,since tumorigenicity is closely associated with the incidence ofprostate cancer, the methods of the present invention may also be usedto arrest metastatic spread from the tumor.

Accordingly, another aspect of the present invention provides a methodof treating prostate cancer cell lines with serum derived from patientson pomegranate juice treatment. The method comprises the step ofadministering to the patient a composition comprising an amount of anextract from pomegranate which is therapeutically effective to increaseapoptosis and/or decrease cell proliferation of cancer cells. The term“therapeutically effective” as used herein means that the amount of theextract of the present invention contained in the compositionadministered is of sufficient quantity present in serum to affectapoptosis and proliferation of cancer cells.

The following examples are intended to illustrate, but not to limit, thescope of the invention. Indeed, those of ordinary skill in the art canreadily envision and produce further embodiments, based on the teachingsherein, without undue experimentation.

EXAMPLES Methods

Preparation of Pomegranate Juice

Pomegranates (Punica granatum, wonderful variety) were picked by hand,washed, chilled to 32° F., and stored in tanks. Then the fruit wascrushed, squeezed, and enzymatically treated with pectinase to yield thepomegranate juice and the by-products, which include the inner and outerpeels and the seeds. Pectinase hydrolyzes alpha-1,4 galacturonidasebonds in pectin and, thus, it improves extraction and filtration, andprevents the formation of pectin gels. The juice was filtered,pasteurized, concentrated, and stored at −18° C.

For this purpose, the pomegranate juice was lyophilized to remove theaqueous part to yield pomegranate juice concentrate. Each day during thestudy period, the pomegranate juice concentrate is diluted 1:5 (v:v)with water in order to obtain a single strength pomegranate juice. Totalpolyphenols concentration in pomegranate juice was determinedspectrophotometrically with the phosphomolybdic phosphotungstic acidreagents. It is also possible to use other methods of juice preparationthat include extract with higher levels of polyphenols.

Design of the Study and Clinical Endpoints

The 2 year, single center, phase II clinical trial was performed todetermine the clinical effect of pomegranate juice on subjects withprostate cancer. The study was fully accrued to 48 subjects in Simontwo-stage after efficacy criteria were met based on a 20% response rate,an alpha of 5%, and 90% power. Clinical endpoints included safety,effect on serum PSA, and exploratory laboratory studies. Patients weretreated with 8 ounces of pomegranate juice by mouth daily, equivalent to1.5 mmol of total polyphenols per day.

Human Studies

Eligible subjects had prior prostatectomy or radiotherapy for primaryadenocarcinoma, rising serum PSA greater than 0.2 ng/ml and less than 5ng/ml, and histological confirmed prostate biopsy showing a Gleasonscore of 7 or less. Just prior to and during the pomegranate juicetreatment, the subjects were followed in 3 month intervals for serumPSA, and blood and urine were collected for laboratory studies. Fastingblood samples were obtained from the subjects in the morning, allowed toclot, and then were centrifuged to obtain serum. The serum was frozenand stored at −80° C. until used for analyses. Prior to the pomegranatejuice treatment, rising serum PSA levels of the subjects were documentedby serial PSA measurement of serum samples of at least one week betweeneach serum sample collection. Safety and tolerability were monitored viacontinuous adverse event reporting.

Cell Culture

Androgen-dependent LNCaP prostate adenocarcinoma were obtained fromAmerican Type Culture Collection (ATCC, Manassas, Va.). The cells weregrown in 75-cm2 flasks (Falcon Primaria, Bedford, Mass.) in RPMI-1640medium without phenol red and supplemented with 10% FBS, 200 IUpenicillin, 200 mg/ml streptomycin, and 4 nM L-glutamine (OmegaScientific, Tarzana, Calif.). The cultures were maintained at 37° C. andsupplemented with 5% CO2 in a humidified incubator. Cells were passagedroutinely at 80% confluence, and fresh medium was replaced every thirdday. All cells used in the study were from the 10^(th) to the 30^(th)passage.

Prostate-Specific Antigen Assays

Serial measurements of serum samples were analyzed for PSA by using theImmulite third generation PSA assay by Diagnostic Products Corporation,Los Angeles, Calif. Three times or more serial serum PSA levels weremeasured at an interval of 3 month. The overall PSADT was estimatedbased on the slope of the linear regression analysis that best fit allavailable PSA values and computed the doubling time as log 2 divided bythe slope.

Analysis of Apoptosis

Induction of apoptosis of LNCaP cells were measured by staining cellswith FITC-labeled annexin V. These cells were cultured either with orwithout serums from patients pretreatment and posttreatment. After 96hours incubation, cells were harvested and externalizedphosphatidylserine at the outer leaflet of the plasma membrane wasdetected using annexin V-apoptosis detection kit according to themanufacture's instruction (Pharmingen, Inc., San Diego, Calif.).

Proliferation Assay

Cells in the Falcon flasks were detached with 0.25% Trypsin-EDTAsolution (Sigma Chemical), centrifuged at 3,000 g for 5 min at 10° C.,and resuspended in fresh medium. Cell viability was assessed via Trypanblue exclusion. Cell growth assays were initiated by seeding cells into96-well plates (Falcon, 53 3072) at various densities in 150 μL culturemedium/well (LNCaP 1000 cells/well, PC-3 2000 cells/well). The cellswere then incubated for 24 hours at 37° C. to allow attachment. Afterattachment, medium and non-attached cells were aspirated and replacedwith 100 μL fresh culture medium with the same supplementation ofantibiotics as indicated before. Growth of cultures was continued foranother 24 hours. Then, 50 μL of pre and post treatment serum were addedand culture growth continued for 96 hours. Control cells receivedRMPI-1640 medium only. Cell growth was determined by CellTiter 96AQassay (Promega, Madison, Wis.).

Urine Sample Analysis

Forty milliliters of urine sample, pomegranate juice pretreatment andposttreatment, was filtered through a Sep-Pak solid phase extractioncartridge (a reverse phase C-18 cartridge; Waters Millipore, UnitedStates). The cartridges were previously activated with 10 mL of MeOH and10 mL of water. Then, the cartridge was washed with 10 mL of water, andthe polyphenols fraction was eluted from the cartridge with 2 mL ofMeOH. A sample of 100 μL of the methanolic fraction was analyzed byliquid chromatography-tandem mass spectrometry.

The high-performance liquid chromatography (HPLC) system equipped withboth a photodiode array detector and a mass detector in series consistedof a HPLC binary pump, autosampler, and degasser controlled by softwarefrom Agilent Technologies (Waldbronn, Germany). The mass detector was anion-trap mass spectrometer (Agilent) equipped with an electrosprayionization (ESI) system (capillary voltage, 4 kV; dry temperature, 350°C.) for the analysis of pomegranate polyphenols in urine samples. Themass detector was equipped with an atmospheric pressure chemicalionization (APCI) system (capillary voltage, 4 kV; dry temperature, 350°C.; crown voltage, 4 kA; APCI temperature, 375° C.).

Statistics

The ANOVA test was performed for all statistical analyses. Results aregiven as the mean±SEM. Assays in each sample were performed intriplicate. All comparisons are shown for data after pomegranate juicetreatment vs. the results obtained before treatment.

Results

There were no serious adverse events reported, and no subjects withdrewbecause of adverse events. The subjects reported that the pomegranatejuice treatment was well tolerated.

Mean PSA doubling time significantly increased with treatment, from amean of 14 to 26 months (p<0.048). The slope of the mean log PSAdecreased from 0.08 to 0.04 on treatment (p<0.019). The result ofchanges in PSADT pre and post treatment of a subject is summarized inFIG. 1. About 31% of patients achieved a decreased PSA level, with dropsranging from 5% to 85%. About 82.5% of patient achieved a lengthened PSAdoubling time.

In vitro assays on the growth of LNCaP showed decreased cellproliferation and increased apoptosis (p<0.07) for post treatmentpatient serum. FIG. 2 shows the effect of pre and post treatment serumon growth inhibition of LNCaP cells. Table 1 shows the effect pre andpost treatment serum on apoptosis of LNCaP cells.

Pomegranate polyphenols were detected of all participants by LC-MS. FIG.3 shows LC-MS analysis of urines, pre and post treatment, obtained froma patient orally administered with pomegranate juice. The relativeintensities are given by the figures in the top right hand corner ofeach chromatogram.

No patients developed metastatic disease on study.

DISCUSSION

Preclinical and animal data suggest that the pomegranate juice canmodulate the growth and progression of prostate cancer. In thepomegranate juice study, the positive and significant beneficial effectson prostate specific antigen (PSA) parameters achieved, coupled withcorresponding laboratory effects of post treatment patient serum onprostate cancer in vitro cell growth and apoptosis. The study detectedpomegranate polyphenols in urine, which suggest that pomegranate juiceis readily absorbed by the body and processed through kidneys.

Laboratory tests showed that serum of patients in vitro reduced cancercell proliferation and increased cancer cell apoptosis as well. Althoughit did not reach statistical significance, in vitro assays using pre andpost treatment patient serum on the growth of lymph node prostate cancercell line (LNCaP) also showed decreased cell proliferation and increasedapoptosis (P<0.07). This is a highly encouraging result for a substancewith no toxicity as this may indicate pomegranate juice dosage be safelyescalated. Albrecht et al. study observed that pomegranate juicefractions acutely inhibited in vitro proliferation of LNCaP, PC-3, andDU145 human cancer cell lines (Error! Reference source not found.). Itshould be noted that dosages used in Albrecht et al. study for thepomegranate fractions may be higher than used in the pomegranate juicestudy.

The study was seeking a signal of efficacy for the intervention, andthese results activated that signal:

About 71% of the patients had stable disease.

About 31% of patients achieved a decreased PSA level.

About 82.5% of patient achieved a lengthened PSA doubling time.

Doubling time prior to starting therapy averaged about 14 months. Aftertreatment with pomegranate juice the doubling time averaged 26 months,an indication that disease progression had slowed. The change indoubling time was significant at the p=0.0001 level. The slowdown indoubling indicates that the threshold for reaching a level at whichmetastases could be expected might be extended for severalyears—possibly not occurring before the patients died of other causes.

The present invention may be embodied in other specific forms withoutdeparting from its essential characteristics. The described embodimentis to be considered in all respects only as illustrative and not asrestrictive. The scope of the invention is, therefore, indicated by theappended claims rather than by the foregoing description. All changeswhich come within the meaning and range of the equivalence of the claimsare to be embraced within their scope.

1. A method of increasing a prostate specific antigen doubling time intreating a person with prostate cancer by dispensing using a solidextract in vivo prepared via a process comprising: crushing andsqueezing whole pomegranates including inner and outer peels and seedsof said whole pomegranates to yield a juice component and an insolubleby-product component; separating said juice component from saidinsoluble by-product component and removing said insoluble by-productcomponent from said juice component to produce a liquid extract fromsaid juice component and a solid extract from said insoluble by-productcomponent, and administering distributing to said a person a compositioncomprising a therapeutically effective amount of said solid extractwherein said composition increases a prostate specific antigen doublingtime.
 2. The method of claim 1 further comprising: administering to saidperson said composition; and, measuring a prostate specific antigendoubling time to confirm a slowing of said prostate specific antigendoubling time.